The long term objectives of the investigator are to understand the precise regulation of cyclic nucleotide synthesis and their function in normal tissues and various disease states. The work proposed will emphasis the regulation and role of cyclic GMP. Model systems will be developed to examine hormonal and E. Coli heat-stable enterotoxin regulation of guanylate cyclase activity and cyclic GMP accumulation in both intact cell and cell-free systems. The mechanisms of coupling of E. Coli heat-stable enterotoxin binding with cyclic GMP synthesis intestinal mucosa and the effects of endothelial dependent vasodilators on vascular smooth muscle cyclic GMP synthesis will be used as example of two model systems for guanylate cyclase regulation. Furthermore experiments will be conducted to reconstitute hormonal regulation of guanylate cyclase in cell-free systems using model cell culture lines and tissues. In addition red cell ghosts will be loaded with various macromolecules involved in cyclic nucleotide synthesis, hydrolysis and function. These loaded ghosts will be fused to cell lines and tissues of interest to modify cyclic nucleotide metabolism and function in order to assess their role in various processes and/or phosphorylation events. Thus, several approaches will be conducted simultaneously to provide new techniques and information to examine the regulation of cyclic nucleotide formation and action.
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